The invention discloses a kit and a method for precise quantification of next-generation sequencing libraries different in GC content by qPCR (quantitative polymerase chain reaction). The kit for precise quantification of the next-generation sequencing libraries different in GC content by qPCR comprises a plurality of standard samples different in GC content, library dilution liquid, qPCR premixed liquid, primers and ddH2O, wherein nucleotide sequences of the primers are as shown in SEQ ID NO:1 and SEQ ID NO:2. The invention further provides the method of adopting the kit for precise quantification of the next-generation sequencing libraries different in GC content by qPCR. The kit and the method have advantages of precise quantification, convenience in use, wide application range, high selectivity, high sensitivity, high specificity and high stability and provide a guarantee for high-quality high-efficiency sequencing.